Reagent formulation for uric acid assay

ABSTRACT

An enzyme reagent formulation for use in assaying a biological specimen for uric acid is prepared as a granular, water-soluble, anhydrous, storage-stable mixture containing uricase, potassium chloride, mannitol, gum acacia, bovine serum albumin, glycine, sodium carbonate and a nitrogen containing polyoxyalkylene nonionic surfactant obtained by the sequential reaction of ethylenediamine with propylene oxide and ethylene oxide in the presence of a catalyst. The surfactant contains polyoxypropylene chains having an average molecular weight of between about 750 and about 6750, and polyoxyethylene chains constituting between about 10 and about 80 weight percent of the surfactant. The surfactant has an advantageous effect upon granulation, dissolution and storage stability of the reagent formulation.

United States Patent [1 1 Monte et al. Dec. 23, 1975 [5 1 REAGENTFORMULATION FOR URIC ACID 3.677.903 1/1912 Bittner 195/1015 R 3,858,8541/1975 Win at al. 195/63 x ASSAY [75] Inventors: Alexander A. Monte;Ching Chiang,

both of Glendora, Calif.

[73] Assignee: Mallineknwt, Inc, St. Louis, Mo.

[22} Filed: May 6, 1974 [21] Appl. No.: 467,431

Related US. Application Data [60] Division of Ser. No. 200,552, Nov. 19,1971, Pat. No 3,816,262, which is a continuationin-part of Ser. No.190,883, Oct. 20, 1971, abandoned.

Primary ExaminerDavid M. Naff Attorney, Agent, or Firm-Mathew D. Madsen[57] ABSTRACT An enzyme reagent formulation for use in assaying abiological specimen for uric acid is prepared as a granular,water-soluble, anhydrous, storage-stable mixture containing uricase,potassium chloride, mannitol, gum acacia, bovine serum albumin, glycine,sodium carbonate and a nitrogen containing polyoxyalkylene nonionicsurfactant obtained by the sequential reaction of ethylenediamine withpropylene oxide and ethylene oxide in the presence of a catalyst. Thesurfactant contains polyoxypropylene chains having an average molecularweight of between about 750 and about 6750, and polyoxyethylene chainsconstituting between about 10 and about 80 weight percent of thesurfactant. The surfactant has an advantageous efl'ect upon granulation,dissolution and storage stability of the reagent formulation.

5 Claims, 1 Drawing Figure REAGENT FORMULATION FOR URIC ACID ASSAYCROSS-REFERENCE TO RELATED APPLICATION cation Ser. No. 190,883, filedOct. 20, l97 l now abanl0 doned.

BACKGROUND OF THE INVENTION This invention relates to the field ofclinical diagnostic testing and more particularly to novel reagents andmethods for making biological assays on body fluids.

A large variety of test reagents and methods are available for use indetermining the character of various body fluids to assist in thediagnosis of certain pathological conditions. Tests for determination ofcertain types of biological activity or the presence and quantity ofcertain biologically active components provide information indicatingthe presence or absence of disease or other physiological disorder. Inaccordance with such tests, the biological specimen to be analyzed, forexample, a sample of a body fluid, is typically mixed with a liquidreagent formulation which contains a reagent capable of effecting areaction which causes a measurable change in the specimen/reagentsystem. Very often the reaction which takes place in the test is anenzymatic reaction. Certain tests are designed, in fact, to determinethe presence of a particular enzyme and in such cases the reagentformulation may contain a substrate upon which the enzyme to bedetermined is known to act. In other cases, the determination may be fora material which is known to be a reactive substrate in an enzymaticallycatalyzed reaction. In either case, the reagent formulation verycommonly contains an enzyme, a coenzyrne or both. Because the catalyticactivity of most enzymes is specific to a particular reaction, testreagents can be formulated which are effective to determine specificbiological components or activities even in a complex body fluidcontaining a large number of other components which might interfere withefforts to obtain a purely chemical analysis. Moreover, many of thecomponents which are to be determined have highly complex chemicalstructures which would render direct chemical analysis difficult even inthe absence of any contaminants.

Unfortunately, enzymes and coenzymes are generally rather delicatematerials which may be readily denatured by heating and which also tendto degenerate upon storage. Many of the substrate materials used inbiological assay reagent formulations are similarly unstable. Liquidreagents containing such components are therefore not generallysusceptible to storage and must be freshly prepared shortly prior to usein clinical diagnostic testing. Because of the relative expense ofenzymes and coenzymes and the skill required to prepare a reagentformulation containing these materials which can be utilized to obtainaccurate clinical diagnostic test results, the instability of the liquidformulations has motivated a substantial amount of research to developreagents in a relatively storage-stable form. Much of this effort hasbeen directed to the development of solid, dry, water-solubleformulations which can be dissolved in water at the time of testing toprovide a fresh liquid reagent useful in the test. Typical 2 prior artdry reagent formulations are disclosed in Deutsch U.S. Pat. No.3,413,198 and Stern et al. US. Pat. No. 3,546,13l.

A dry reagent formulation satisfactory for use in preparing liquidreagents for routine clinical diagnostic tests should satisfy a numberof criteria. It must be readily soluble in a solvent compatible with thebiological specimen, usually water. It should be capable of solubilizingproteinaceous material in the specimen. Moreover, it should be readilysusceptible to packaging in convenient sized packages and be adapted forrapid dissolution in the solvent to provide a liquid reagent of properstrength for a given test or series of tests.

SUMMARY OF THE INVENTION It is an object of the present invention toprovide improved dry, water-soluble, reagent formulations for use inconducting clinical diagnostic tests. It is a further object of thepresent invention to provide such formulations which can be readilygranulated and shipped or stored in granular form. It is a particularobject of the invention to provide such reagent formulations infreeflowing, granular form at consistent bulk densities so that they maybe delivered to a volumetric packaging or tableting operation inconsistent weight amounts. Additional objects of the invention includethe provision of dry reagent formulations having a high capacity forsolubilizing protein; the provision of such formulations having a highdegree of storage stability; the provision of methods for preparing thedry reagent formulations of the invention; and the provision of methodsfor conducting clinical diagnostic tests utilizing such reagentformulations. Other objects and features will be in part apparent and inpart pointed out hereinafter.

In one of its aspects, therefore, the present invention is directed to areagent formulation for use in conducting a clinical diagnostic test ona biological specimen. The reagent formulation comprises a solid,water-soluble, substantially anhydrous, storage-stable mixturecontaining a reagent capable of participating in a test reaction toeffect a measurable change in a test system, and a solidnitrogen-containing polyoxyalkylene nonionic surfactant. The surfactanthas a structure corresponding to that obtained when ethylene diamine isreacted sequentially with propylene oxide and ethylene oxide in thepresence of a catalyst and the polyoxypropylene chains of the surfactanthave an average molecular weight of between about 750 and about 6750.

The invention is further directed to a method of conducting a clinicaldiagnostic test on a biological specimen using the aforementionedreagent formulation. The method comprises dissolving the reagentformulation in water to produce a liquid reagent; mixing the liquidreagent with a specimen to form a specimen/reagent test system; andmeasuring a change in the system resulting from the reaction between thereagent and the specimen.

The invention is also directed to a method of preparing the novelreagent formulation. The method comprises the steps of mixing a reagentcapable of participating in a test reaction to effect a measurablechange in a test system, a nitrogen-containing polyoxyalkylene nonionicsurfactant of the above-noted character, and a solvent for thesurfactant; anad removing the solvent to form a substantially anhydrous,water-soluble, freeflowing, granular solid.

DESCRIPTION OF THE DRAWING The drawing is a grid illustrating themolecular structure of various commercially available nonionicsurfactants useful in the practice of the invention. The coordinates ofeach point on the grid correspond to the chain size of thepolyoxyethylene hydrophile and polyoxypropylene hydrophobe moieties of aparticular surfactant. Boundary lines set out on the grid separate theareas encompassing surfactants which assume different physical states.

DESCRIPTION OF THE PREFERRED EMBODIMENTS To facilitate preparation ofliquid reagents from solid formulations in the clinical laboratory, itis highly desirable to package the solid formulations in proper unitaryamounts. Thus, for example, the solid formulations may be encapsulatedor tabletted with the proper quantity of reagent in each capsule ortablet for conducting a single test. Alternatively, a multi-test packagecan be provided from which the proper amount of liquid reagent isprepared for conducting a specified number of tests.

Where a solid reagent formulation is packaged in unitary amounts,accuracy of metering the solid material into each capsule, tablet ormulti-test package is important. The metering equipment which is usedfor delivering solid materials in packaging and tableting operations,however, almost universally operates on a volumetric basis. Unless thesolid material is free-flowing and has a consistent bulk density,therefore, it cannot be delivered in consistent weight amounts to eachpackage, capsule or tableting station using conventional equipment.

To provide a solid formulation in free-flowing form of consistent bulkdensity, it is preferably granulated prior to packaging. Granulationconverts a powdered material into a material constituted by smallagglomerates of relatively uniformsize. Properly prepared, the granularmaterial is free-flowing, has a consistent bulk density and is readilyhandled by the metering devices used in packaging operations. Togranulate a powdered material, the powder is typically mixed with abinder dissolved in a volatile solvent, wet screened, dried by drivingoff the solvent, and dry screened following the drying step. In additionto the binder, a lubricating substance is normally incorporated in thegranulation mass to further enhance the flow characteristics of thegranules, especially under the compressive stress of tabletingoperations.

As noted, solid formulations useful as reagents for conducting clinicaldiagnostic tests on biological specimens should have certain additionalproperties. Because they are dissolved in water to produce a liquidreagent, all components, including the binder, should be readilywater-soluble. Because many of the tests involve enzymatic reactionsand/or proteinaceous substrates, the formulation should possessdetergent properties for solubilizing protein.

It has now been discovered that the above objectives can be met and thateffective clinical reagent formulations for the determination of certainbiological properties of body fluids can be produced in free-flowing,granular form through the use of particular nitrogenbearingpolyoxyalkylene nonionic surfactants. Test formulations granulated withthe aid of these surfactants are well adapted to precision packaging andtableting operations. Because of their free-flowing character andconsistent bulk density, they can be delivered to either a packaging ortableting operation in consistent weight amounts by volumetric metering.As a consequence, clinical test reagents formulated at a centrallocation remote from a clinical laboratory can be utilized to prepareliquid test reagents for clinical use without the need for weighing,analyzing, or other procedures by the clinical chemist or technician.

The nitrogen-containing surfactants which are useful in the formulationsof the invention possess the unique multiple capability of serving asbinders, lubricants and solubilizers for protein. Moreover, they arethemselves water-soluble, thus promoting the dissolution of the reagentformulations in water to provide clinical liquid reagents. Thesesurfactants are sold under the trade designation Tetronic" by WyandotteChemical Corporation. They are normally prepared by sequential reactionof first propylene oxide and then ethylene oxide with ethylene diaminein the presence of an alkaline or acid catalyst. Normally thesesurfactants are prepared at elevated temperatures using alkalinecatalysts such as sodium hydroxide, potassium hydroxide, sodiumalkoxide, quarternary ammonium bases and the like. Other methods areavailable for the preparation of these surfactants. The preparation ofsurfactants such as those utilized in the formulations of the inventionis more fully described in U.S. Pat. No. 2,979,528.

The properties and physical state of nonionic surfactants havingstructures corresponding to those derived from ethylene diamine,propylene oxide and ethylene oxide vary with the lengths of thepolyoxypropylene and polyoxyethylene chains. As the drawing shows, thephysical state of these surfactants is largely dependent upon theproportionate weight of the surfactant constituted by thepolyoxyethylene chains, but is also influenced by the average molecularweight of the polyoxypropylene moieties. The polyoxypropylene chains arehydrophobic while the polyoxyethylene chains are hydrophilic. Thus, thesurfactants having polyoxypropylene units of low average molecularweight are more water-soluble than those having polyoxypropylene unitsof a higher average molecular weight. The numbers set out on the face ofthe grid correspond to particular members of the Tetronic series. Eachnumber is located at a point on the grid whose coordinates correspond tothe polyoxyethylene and polyoxypropylene chain sizes of the particularproduct which is commercially designated by said number.

Essentially any surfactant whose structure is defined by the coordinantsof a point lying in the grid of the drawing may be utilized in theformulations of the invention. It is preferred, however, that thesurfactant be solid or at least semi-solid. A greater proportion of thesolid surfactants can be satisfactorily incorporated in a reagentformulation and thus a greater binding and lubricating capacity isobtained without adversely affecting other properties of theformulation. Desirably, on the order of 2.5 to 5% by weight of thepreferred solid surfactants are incorporated in the reagent formulations. When the liquid formulations are used, it is not alwayspossible to incorporate more than 2 or 3% by weight of the surfactantwithout imparting a somewhat waxy character to the formulation. The useof 2 to 3% by weight of a liquid Tetronic surfactant produces a usefulproduct, but the binding and lubricating capabilities of the surfactantare not always fully exploited at such a level. Granules having the mostdesirable propcities are obtained using solid or semisolid surfactants.

Since the dry reagent formulations of the invention are dissolved inwater for use in conducting clinical diagnostic tests, it is alsodesirable that the surfactant component promote the dissolution of thegranular productv Thus, it is preferred that the surfactant be ashydrophilic as possible, i.e., that the molecular weight of thepolyoxypropylene hydrophobe moiety of the surfactant be relatively low.Thus, the preferred surfactants for use in the formulations of theinvention are those which are both solid or semi-solid in physical stateand relatively hydrophilic. Solidstate surfactants with polyoxypropylenechains having an average molecular weight of less than about 4000 areespecially preferred, with the most suitable surfactants being thosewhose polyoxypropylene chains have an average molecular weight ofbetween about 2750 and about 3750 and whose weight percentage ofpolyoxyethylene units is between about 70% and about 80%. Two particularsurfactants whose weight and structure characteristics fall within thelatter limits are those sold under the trade designations Tetronic 707and Tetronic 908. Tetronic 707" has a polyoxypropylene hydrophobemolecular weight on the order of 2750 and a weight percentage ofpolyoxyethylene units of about 70 while Tetronic 908 has apolyoxypropylene molecular weight of about 3750 and a weight percentageof polyoxyethylene units of about 80%. Good results are also obtainedwith surfactants whose polyoxypropylene chains have an average molecularweight of between 750 and 4000 with a weight percentage of between about35% and about 65% polyoxyethylene units. Other surfactants within thegrid of the drawing are reasonably satisfactory but less effective thanthose represented by the right lower comer of the grid.

ln addition to their advantageous effect upon granulation anddissolution of dry clinical test reagent formulations, surfactants ofthe above-noted character have been found to be effective forsolubilizing protein. As indicated above, this is a highly advantageouscharacteristic, since enzymes and other proteinaceous matter derivedfrom either the reagent formulation or the specimen commonly participatein the test reactions. By solubili zing protein, the surfactantsfunction to facilitate the progress of the test reaction and thusenhance the effectiveness of the reagent formulation. It may, therefore,be seen that incorporation of these surfactants in clinical testformulations uniquely provides multiple advantages in the preparation,packaging, dissolution and functional operation of clinical reagentformulations.

It has further been discovered that the dry clinical reagentformulations of the invention are quite stable and generally possessgood shelf life characteristics. Although we cannot precisely accountfor the particular ingredient or combination of ingredients whichimparts the high degree of storage stability, it appears that suchstability may be a somewhat general characteristic of dry clinicalreagent formulations which include the particular nitrogen-containingnonionic surfactants used in our formulations. If so, the ability toimpart storage-stability represents a further aspect of the uniquemultiple function of this type of surfactant in such formulations.

To prepare the reagent formulations of the invention, the surfactant ismixed with a volatile solvent and at least one reagent capable ofparticipating in a test rcaction to effect a measurable change in areagent/specimen test system. The surfactant should be soluble up to theamount present in the solvent which is utilized. Solvents which may beused include methylene chloride, chloroform. methanol. benzene. water,methanol/water, and chloroform/methylene chloride. After thorough mixingand appropriate size classification, the solvent is removed to yield agranular product.

In a preferred embodiment of the invention, the ingredients of theformulation, in dry particulate form, are thoroughly blended in amechanical mixer. With the mixer running, a granulating solutioncontaining the solvent and the surfactant, preferably that sold underthe trade designation Tetronic 707 or Tetronic 908, is added. Additionalsolvent is used as needed to produce granular agglomerates of thedesired size and wetness.

The resulting wet granulation is screened through a coarse screen, forexample 10 mesh, then spread in thin layers in trays and dried atreduced pressure, for example, 25 inches Hg absolute or less. Dependingon the heat sensitivity of the formulation, drying is normally carriedout at room temperature or at modest elevated temperature (up to about37C.). Generally, the depth of the wet granules in the trays should notexceed about V2 inch to 4 inch.

After completion of the drying cycle, the dried granulation isrescreened through a finer screen, for example, 20 to 30 mesh, blendedthoroughly and packaged in containers essentially impervious tomoisture. Since the components of the reagent formulation are frequentlymoisture sensitive, the formulation should not be exposed to a relativehumidity of more than about 5% after removal from the dryer.

The reagent formulations of the invention are adapted to be packaged insmall unitary packages. For example, sufficient reagent formulation fora single assay may be tabletted or packaged in a capsule. The reagentformulations are also adapted to packaging in such containers as foilstrip packets, utilizing automatic packaging machinery. Utilizing thispackaging approach, sufficient reagent formulation to carry out asuitable predetermined number of tests, such as 10, 25, or 50 tests, maybe accurately packaged in a single foil packet. The user then simplydissolves the contents of the multiple test packet in a predeterminedvolume of water and uses a suitable aliquot of the resulting liquidreagent in the preformance of each of a series of assays for the desiredbiological substance or property.

In some instances, depending on the nature of the components and theircompatibility, all of the reagents necessary in a single assay ordetermination may be included in a single formulation. ln otherinstances, incompatabilities and/or other considerations may make itdesirable to segregate certain reagents in which case two or morereagent formulations are prepared in accordance with the invention.

To conduct a clinical diagnostic test using the formulations of theinvention, the liquid reagent produced by dissolving the dry formulationin a predetermined amount of water is mixed with the biological specimenin a predetermined volumetric or weight ratio. With the aid ofappropriate instrumentation as required, the resulting specimen/reagentsystem is observed for the presence, absence, nature and extent of aphysical, chemical or biological change. Such change as does occur ismeasured to provide the desired information for use in the clinicaldiagnosis.

Exemplary reagent formulations prepared in accordance with the inventionand useful for the determination of hemoglobin, blood urea nitrogen,total protein, serum glutamic oxaloacetic transaminasc, alkalinephosphatase, glucose. inorganic phosphorus. lactate dehydrogenase-L,serum glutamic pyruvic transami nase, uric acid (colorimetric) and uricacid (u.v.) are set forth in Table l. The preferred compositions ofthese reagent formulations and methods for preparing them are describedin the examples following Table 1 which more fully illustrate theinvention.

Table l Exemplary Clinical Test Reagent Formulations GranulatingSolution Poly- No. of Dry Ingredients ethylene Theo- Tests Formu- Typeof Formu (Reagents, Etc.) TETRONIC glycol CH Cl rctical (Thoulationlation Name/Formula Wt. (g.) 707 (g.) 6000 (g) (ml.) I) Yield (g.)sands) A Reagent Formu- NaHCO 300) 30 (a) 200 1000 50 lation for Hemo-K,-,Fe(CN),; 50) (h) 300 globin Assay KCN 30) Mannitol 590) T EnzymeFormu- Uricasc Formulation for Uric lation (equiva- Acid Assay lent to0.05

units) Glycine H315) 4.50 (a) 25 7.5 Sodium carbonate. anhydrous 39.75)(h) U Copper Reagent Tris-(hydroxy- Formulation for methyl)-amino- UricAcid Assay methane H150) 7.05 (a) [5 234 7.5

Sodium Bicar bonate l 12.50) (b) 5 Copper Sulfate, anhydrous L95) VNeocuproine Formu- Neocuproine HCl 5.25) 4.75 (a) 30 I60 7.5

lation for Uric Renex 35 150 (b) )0 Acid Assay W Blank Formu- UricasePlacebo 19.50) 4.50 (a) 177 7.5

lation for Uric Glycine l 13.25 Acid Assay Sodium carbonate,

anhydrous 39.75

1 l (:1) indicates amount of CH.,CI used as carrier for Tetronic 707 (b)indicates amount of additional CH,Clused to optimize granulation EXAMPLE1 Hemoglobin Reagent Formulation and Assay Composition of the reagentformulation useful for hemoglobin assay is set forth as formulation A inTable I.

To prepare this formulation, sodium bicarbonate (300 g), milledpotassium ferricyanide (50 g.) and potassium cyanide g.) were initiallyadded to 21 Hobart bowl and mixed with a stainless steel spatula.Mannitol (590 g.) was then added and the resulting blend was agitatedfor five minutes in the mixer. While agitation was continued, a solutionof Tetronic 707 (30 g.) in methylene chloride (200 ml.) was added. Anadditional amount of methylene chloride (300 ml.) was then added toproduce the proper granulation.

The wet granulation was screened through a No. 10 mesh stainless steelscreen and the wet screened material was transferred to 8 inches X 12inches Pyrex drying trays, at a depth of between about /2 inch and about54 inches in each tray. The granulation was then dried in a vacuum ovenfor 15 hours at a temperature of C. and a pressure of 25 inches Hg.

The dried granulation was removed from the vacuum oven in an environmentwhere the relative humidity was not more than 5%. The dried granulationwas then screened through a No. 20 mesh stainless steel screen using anErweka oscillator. The screened, dried granuof the dissolved reagent,erythocytes in the blood are hemolyzed releasing hemoglobin which isoxidized to methemoglobin. Methemoglobin is converted tocyanmethemoglobin whose formation alters the optical density of thereagent/specimen system. The optical density of the reagent/specimensystem is measured at 540 nm. using a suitable spectrophotometer andcompared against a reagent blank set at transmission. The hemoglobinlevel is then determined by reference to a standard curve.

To prepare a liquid reagent sufficient for 50 tests, formulation A (1.00g.) is dissolved in distilled water and the resulting solution isdiluted to 250 ml. and mixed thoroughly. The reagent solution thusproduced is stable for three months at room temperature if protectedfrom light.

To conduct the hemoglobin assay test, a reagent/- specimen test systemis prepared by adding 20 microliters of well mixed blood (collected withan anticoagulant) to 5 ml. of the above solution of formulation A in aclean test tube. The contents of the tube are mixed thoroughly andallowed to stand at room temperature for at least five minutes. Theoptical density is then measured as described above to determine thehemoglobin level.

EXAMPLE 2 Colorimetric Formulations and Assay for Uric Acid For thecolorimetric uric acid test, three separate formulations are provided.These three formulations are set forth in Table l as formulations T, Uand V. Predetermined amounts of these formulations are dissolved inseparate portions of water to provide liquid reagents for use in makingthe uric acid assay.

Preparatory to blending the constituents of enzyme reagent formulationT, the modified uricase component thereof was produced. To produce themodified uricase, a borate buffer was initially prepared by dissolvingboric acid (50 g.) in distilled water (3.5 l.) and titrating theresulting solution to a pH of 9 with 10% solution of sodium hydroxide.The titrated solution was then diluted to a total volume of 4 l. (0.2 Min borate) and chilled in refrigerator prior to use. Uricase (about 40mg.) was transferred to a 250 ml. beaker by streams of the borate bufferdelivered from a wash bottle. The uricase used was uricase solution in50% glycerol obtained from Boehringer Mannheim Corporation (Cat. No.15074 EVAC) and having a specific activity of about 4.5 U/mg. at 25C.and about 10.5 U/mg. at 37C. After the uricase was transferred,additional borate buffer was added to bring the total volume of uricasesolution in the beaker to approximately 100 ml. The diluted uricasesolution was then dialyzed against approximately 2 l. of 0.2 M boratebuffer for 4 hours, contaminated buffer being replaced with fresh bufferat the end of the first 2 hours of dialysis. While dialysis was inprogress potassium chloride (6 g.), mannitol 4 g.) and gum acacia (4 g.)were dissolved in 0.2 M borate buffer (100 ml.). The resulting solutionwas clarilied by centrifugation at 10,000 rpm and 10C. for 16 minutesusing a No. 872 angle rotor in an [EC B-20 refrigerated centrifuge.Bovine serum albumin (0.4 g.) and approximately 67,200 units of catalasewere added to the clarified solution to produce a solution referred tohereinafter as the inert solution.

After dialysis of the uricase solution was complete, the dialyzeduricase solution was transferred to a 500 ml. beaker and combined with a200 ml. portion of the inert solution. Distilled water (200 ml.) wasthen added and the resulting solution was thoroughly mixed. Thissolution was then transferred into twoseparate freezedrying vessels andshell frozen in a dry ice/alcohol bath at a temperature of 60C. orbelow. The frozen solution was lyophilized at -60C. to -70C. and anabsolute pressure of millimicrons mercury for 2024 hours. The resultantlyophilized powder was collected under an atmosphere having a relativehumidity of less than 5% and stored in a dessicator at 4C. Approximately19.5 g. of modified uricase was obtained.

To prepare uricase reagent T, modified uricase (equivalent to 0.05units/test). glycine l 13.25 g.) and anhydrous sodium carbonate(39.75g.) were blended in a Hobart bowl and thoroughly agitated topromote intimate mixing. With the mixer running, a solution of Tetronic707 (4.50 g.) in methylene chloride (25 ml.) was introduced. Additionalmethylene chloride (approximately ml.) was subsequently added to producethe desired degree of granulation and wetnessv The wet granulation wasthen screened and dried and the resulting dry granulation rescreened andpackaged in the manner described in Example 1 for hemoglobin reagentformulation A.

In the preparation of copper reagent formulation U,tris-(hydroxymethyl)-aminomethane (112.50 g.), sodium bicarbonate (112.5g.) and anhydrous cupric sulfate (1.95 g.) were blended in a Hobart bowland agitated to promote intimate mixing. With the mixer running, asolution of Tetronic 707 (7.05 g.) in methylene chloride (15 ml.) wasadded. Additional methylene chloride (approximately 5 ml.) wassubsequently introduced to produce the desired degree of granulation andwetness. The wet granulation was then screened and dried and theresulting dry granulation rescreened and packaged in the mannerdescribed in Example 1 for hemoglobin reagent formulation A.

To prepare neocuproine reagent formulation V, neocuproine hydrochloride(5.25 g.), Renex-35" 150 g.) and Tetronic 707 (4.75 g.) were blended in21 Hobart bowl and thoroughly agitated to promote intimate mixing.Methylene chloride (approximately 40 ml.) was then introduced to producethe desired degree of granulation and wetness. The wet granulation wasthen screened and dried and the resulting dry granulation rescreened andpackaged in the manner described in Example 1 for hemoglobin reagentformulation A.

Dissolved in separate portions of water, formulations T, U and V provideliquid reagents useful in assaying a biological specimen for uric acid.Uric acid in the specimen reduces cupric ion of formulation U to cuprousion which in turn reacts with neocuproine of formulation V in bufferedsolution to form a color complex. The resulting optical density of thetest system is compared with the optical density of a blank prepared inthe same manner as the test system but further including uricase fromformulation T which destroys uric acid. The differences in absorbancesbetween the test system and the blank is proportional to the serum uricacid content.

To prepare the liquid enzymatic reagent, formulation T (1.18 g.) isdissolved in distilled water (150 ml.). A uricase blank solution(formulation W) is prepared by dissolving (1.18 g.) in distilled water(150 ml.). The copper-bearing liquid reagent is prepared by dissolvingformulation U (1.55 g.) in distilled water (25 ml.). Aneocuproine-bearing liquid reagent is prepared by dissolving formulationV (1.06 g.) in distilled water (25 ml). The liquid reagent solutions offormulations U and V are stable indefinitely at room temperature whilethe solution of formulation T should be prepared fresh daily. Theresulting solutions are sufiicient for conducting 50 tests.

In the conduct of the test, a 3 ml. portion of the solution offormulation T is added to one test tube and 3 ml. portion of formulationW is added to a second test tube. 0.1 ml. of serum is then added to bothtubes to provide a specimen/reagent test system in the tube containingdistilled water and a blank test system in the tube containing thesolution of formulation T. The contents of both test tubes are thenincubated for 15 minutes at 37C., following which 1 ml. of a combinedcolor reagent mixture, prepared by mixing equal volumes of the solutionsof formulations U and V, is added to both the specimen/reagent testsystem and the blank test system. Both of the test systems are allowedto stand at room temperature for 15 minutes after addition of thecombined color reagent mixture and the light absorbance of each systemis then measured at 455 nm on a spectrophotometer set at transmis sionon a water blank. To provide the data required for the calculation ofuric acid in the serum. another optical density measurement is taken ona standard reagent blank. The standard reagent blank is prepared byadding uric acid l mg.) and lithium carbonate (60 mg.) to distilledwater (about 500 ml.) and warming the mixture to 60C. to dissolve theadditives. The resulting solution is cooled to room temperature anddiluted to a total volume of l000 ml. with additional quantities ofdistilled water. 3 ml. of this reagent blank is then added to a testtube and processed in the same fashion as the blank and thespecimen/reagent test system including addition of serum, incubation,addition of the abovenoted combined color reagent mixture and a -minutehold prior to measurement of optical density. The mg% uric acid in theserum specimen is then determined in accordance with the followingcalculation:

Test O.D. (vs. water) Blank O.D. (vs. water) Standard O.D. (vs. reagentblank) x 10mg.

Two formulations are used in the uric acid (U.V.) test. One of theseformulations is formulation T of Example 10 while the other is set forthin Table l as formulation W. Predetermined amounts of these formulationsare dissolved in separate portions of water to provide liquid reagentsfor use in making the uric acid (U.V.) assay.

in preparing formulation W, a uricase placebo is used. This is preparedin the same manner as the modifled uricase component of formulation T asdescribed in Example If) except that the uricase is omitted.

To prepare formulation W, uricase placebo (19.50 g.), glycine 1 I325 g.)and anhydrous sodium carbonate (39.75 g.) were blended in a Hobart bowland thoroughly agitated to promote intimate mixing. With the mixerrunning, a solution 'of Tetronic 707 (4.50 g.) in methylene chloride (25ml.) was introduced. Additional methylene chloride (approximately 10ml.) was subsequently added to provide the desired degree of granulationand wetness. The wet granulation was screened and dried and theresultant dried granulation rescreened and packaged in the mannerdescribed in Example 1 for hemoglobin reagent formulation A.

A liquid reagent solution of formulation W is used in conjunction with aliquid reagent solution of formulation T in practicing the uric acid(U.V.) test. In the presence of the uricase of formulation T, uric acidfrom the specimen reacts with water and oxygen to form allantoin, carbondioxide, and hydrogen peroxide. Light absorbance at 293 nm, theabsorption peak of uric acid, is measured before and after treatment ofthe specimen with uricase from formulation T with the difference inabsorbance being proportional to the uric acid present in the system.Allantoin, the product of the uricase catalyzed reaction of water, uricacid and oxygen, does not absorb at 293 nm.

The liquid reagent solution of formulation T is prepared as described inExample l0 above. To prepare a blank liquid reagent, formulation W (1.18g.) is dissolved in distilled water 9(ml.). As noted above, the solutionof formulation T should be prepared fresh daily. The liquid reagentsolution of formulation W is stable for 1 month when refrigerated. Theresulting solution is sufficient for conducting 50 tests.

In conducting the test, a blank system is prepared by mixing thesolution of formulation W (3.0 ml.) with a specimen of serum (100 pl)while a specimen/reagent test system is prepared by mixing the solutionof formulation T (3.0 ml.) with a specimen of the same serum [00 #1).Both the blank system and the specimen/reagent test system are incubatedat 37C. for 15 minutes. The incubated mixtures are then transferred tocuvettes of a spectrophotometer having a l centimeter light path. Theinstrument is zeroed at 0.800 O.D. with the blank at 293 nm. Theabsorbance of the unknown is then read and the mg% uric acid in thespecimen determined in accordance with the following calculatlOnI (0.8O.D. of unknown) X 4|.36

In view of the above, it will be seen that the several objects of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above methods and productswithout departing from the scope of the invention, it is intended thatall matter contained in the above description shall be interpreted asillustrative and not in a limiting sense.

What is claimed is:

I. An enzyme reagent formulation for use in assaying a biologicalspecimen for uric acid comprising a granular, wate r-soluble, anhydrous,storage-stable mixture containing:

a. uricase;

b. potassium chloride;

mannitol; gum acacia; bovine serum albumin; glycine;

sodium carbonate; and

a nitrogen-containing polyoxyalkylene nonionic surfactant obtained bythe sequential reaction of ethylenediamine with propylene oxide andethylene oxide in the presence of a catalyst, said surfactant containingpolyoxypropylene chains having an average molecular weight of betweenabout 750 and 6750 and polyoxyethylene chains constituting between about10 and about weight percent of said surfactant.

2. A reagent formulation as set forth in claim 1 wherein saidnitrogen-containing surfactant is solid and the polyoxypropylene chainsthereof have an average molecular weight of less than about 4000.

3. The method of preparing a granular, water-soluble, free-flowing,substantially anhydrous, storage-sta ble enzyme reagent formulation foruse in assaying a biological specimen for uric acid, said methodcomprising the steps of:

l. preparing a mixture containing:

a. uricase;

b. potassium chloride;

c. mannitol;

d. gum acacia;

e. bovine serum albumin;

f. glycine;

g. sodium carbonate;

h. a nitrogen-containing polyoxyalkylene nonionic surfactant obtained bythe sequential reaction of ethylenediamine with propylene oxide andethylene oxide in the presence of a catalyst, said surfactant containingpolyoxypropylene chains having an average molecular weight of betweenabout 750 and about 6750 and polyoxyethylene 14 propylene chains thereofhave an average molecular weight of less than about 4000.

S. The method as set forth in claim 4 wherein the polyoxypropylenechains of the surfactant have an average molecular Weight of about 2750and the weight percentage of the polyoxyethylene units thereof is aboutUNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTIONPATENT NO. 2 3,928,137 DATED I December 23, 1975 INVENTORt'S) IAlexander A Monte: Ching Chiang, Both of Glendora, California It iscertified that error appears in the above-identified patent and thatsaid Letters Patent are hereby corrected as shown below:

In col. 2, line 66. "anad" should read and In col. 6, line 48,"preformance" should read performance In columns 7 and 8, Table l, thefirst two lines of the text relating to Formulation W should read asfollows:

- W Blank Formu- Uricase Placebo 19.50) 4.50 (a) 25 177 7.5

lation for Uric Glycine 113.25 (b) 10 In col. 11, line 6 "9(ml.)" shouldread (150 ml.

In col. l2 (Claim 1), line 42. "and 6750" should read and about 6750Signed and Sealed this twentieth D3) Of ApriI1976 [SEAL] AIIESI.

RUTH C. M ASON C. MARSHALL DANN Alluring ()jfr'ter ('mmnissimwrnj'lau'nls and Trademarks

1. AN ENZYME REAGENT FORMULATING FOR USE IN ASSAYING A BIOLOGICALSPECIMENT FOR URIC ACID COMPRISING A GRANULAR, WATER-SOLUBLE, ANHYDROUS,STORAGE-STABLE MIXTURE CONTAINING: A. URICASE; B. POTASSIUM CHLORIDE; C.MANNITOL; D. GUM ACACIA; E. BOVINE SERUM ALBUMIN; F. GLYCINE; G. SODIUMCARBONATE; AND H. A NITROGEN-CONTAINING POLYOXYALKYLENE NONIONICSURFACTANT OBTAINED BY THE SEQUENTIAL REACTION OF EHYLENDIAMINE WITHPROPYLENE OXIDE AND ETHYLENE OXIDE IN THE PRESENCE OF A CATALYST, ANAVERAGE MOLECULAR WEIGHT OF PROPYLENE CHAINS HAVING AN AVERAGE MOLECULARWEIGHT OF BETWEEN ABOUT 750 AND 6750 AND POLYOXYETHYLENE CHAINSCONSTITUTING BETWEEN ABOUT 10 AND ABOUT 80 WEIGHT PERCENT OF SAIDSURFACTANT.
 2. A reagent formulation as set forth in claim 1 whereinsaid nitrogen-containing surfactant is solid and the polyoxypropylenechains thereof have an average molecular weight of less than about 4000.2. removing the solvent to form a substantially anhydrous, free-flowing,water-soluble, granular solid.
 3. The method of preparing a granular,water-soluble, free-flowing, substantially anhydrous, storage-stableenzyme reagent formulation for use in assaying a biological specimen foruric acid, said method comprising the steps of:
 4. The method as setforth in claim 3 wherein the nitrogen-containing surfactant is solid andthe polyoxypropylene chains thereof have an average molecular weight ofless than about
 4000. 5. The method as set forth in claim 4 wherein thepolyoxypropylene chains of the surfactant have an average molecularweight of about 2750 and the weight percentage of the polyoxyethyleneunits thereof is about 70%.